Imaging Structural Proteins

1. Imaging Structural Proteins Annette Doyle School of Pharmacy & Biomolecular Sciences & General Engineering Research Institute

2. Contents • Introduction –AFM, Fluorescence Microscopy, Cell Structure, Cytoskeleton and Cell Mechanics • Project Rationale • Methodology • Results • Summary & Advantage of AFM

3. Introduction • Understanding how cells function is critical to understanding disease • To understand how cells function requires us to understand how cells are organised structurally at the molecular level • To do this requires the use of imaging technology which enables us to image structures at the sub-nanometre level

4. Atomic Force Microscope • Images collected in intermitting contact mode (tapping mode) • The laser spot is aligned at the end of the cantilever on the opposite side of the tip and deflected onto a photo detector • In tapping mode the cantilever is oscillated at, or near, its resonance frequency at a set-point amplitude • Any changes from the set-point amplitude value of the oscillating cantilever are recorded and the image is constructed • Height and amplitude images are captured as the tip scans the sample

5. Fluorescence Microscopy • A Protein/Cell is stained with a probe specific to the protein of interest with a fluorescent conjugate • The conjugate when subjected to a laser at specific wavelength (excitation) emits light at a different wavelength (emission) • The emitted light rays are recorded and the image constructed • Multiple fluorescent probes can be used, but must ensure no cross over in the excitation or emission wavelengths

6. Cell Structure Intermediate filaments (Green) Double Membrane Microtubules (Blue) Nucleus Actin Cortex (Red)

7. Cytoskeleton • Acts as a ‘skeleton’ providing the cell with shape, mechanical integrity and ability to move. • Consists of 3 types of filamentous protein • Actin • Microtubules • Intermediate Filaments • These extend throughout the cell forming a complex but organised network

8. Cytoskeleton • The filaments interact with each other, the cell membrane, nucleus and cell substrate • It has been suggested that the mechanical integrity of the cells is mainly due to the actin filaments • In healthy cells the cytoskeleton is highly organised • In pathological cell such as cancer cells the cytoskeleton becomes disorganised

9. Cytoskeleton • Fluorescent image of MRC5 human lung fibroblast cells • Actin filaments (green), DNA (blue) • Actin stress fibres present, which play an important role in the cell function

10. Cell Mechanics • Mechanical properties are defined by the cytoskeleton and the volume of a cell • Much of the work is based on understanding the cell mechanics of normal cells and change, if any that occur in pathological cells • Higher levels of protein in the cell may cause more bundling or a change in orientation of the cells or even bigger bundles

11. Cell Mechanics • Changes to the cytoskeleton in the form of bundles may affect the elasticity or ‘stiffness’ of cells • It has been reported that cancer cells are significantly less stiff than healthy cells • Most research is based on a complete cell but we are looking at the organisation and reorganisation of purified actin in the presence of a bundling protein

12. Project Rationale • The proteins that interact and bundle actin cytoskeleton have been shown to have high levels in various cancers • Therefore we aim to determine if these proteins bundle actin and would therefore have an effect on the cytoskeleton's mechanical properties • Today, we will concentrate on the imaging to gain information about the structure and size of filaments

13. Methodology • For initial experiments purified proteins were used • Actin was purified from rabbit muscle and the bundling protein from yeast cells • Actin filaments were imaged in the absence and presence of a bundling protein

14. Mica with Buffer • Mica substrate was imaged to ensure no artefacts are detected from buffer solution used to enable filament formation

15. Actin Filaments Figure 1: 3D height image of actin filaments Figure 2: Height image of unstable filaments – Monomeric actin • Actin filaments were imaged (fig 1) and correspond to the size quoted in the literature 7-8nm • Imaging actin filaments was difficult as they were unstable (fig 2)

16. Actin Filaments with Phalloidin Fig 3: Confocal fluorescent image of actin filaments Fig 4 & 5: AFM 3D height image of actin filaments • Phalloidin stabilises actin filaments • Filament size are slightly larger with phalloidin attached – 8-11nm • Actin monomers form a double helix filament

17. Actin Filaments with Phalloidin Fig 6: 3D height image with phalloidin • Helical structure of actin filaments, size shown by upper graph, major cleft, approx 30nm • Fluorescent conjugate (indicated with arrow) • Size of conjugate measured and shown in lower graph, approx 1.2nm (reported to be between 1.2 -1.5nm)

18. Actin with the bundling protein Fig 8: 3D height image of actin and bundling protein bound along the filament Fig 7: Amplitude image • It can be clearly seen that the bundling protein causes a change in organisation of actin, causing filaments to bundle together

19. Actin with the bundling protein Fig 10: Amplitude image Fig 9: Phase image • Phase imaging (Fig 9) provides nanometre scale information about the surface structure of molecules • Can distinguish between actin filaments and bundling protein • Shows bundling protein bound along the filament • Amplitude image (Fig 10) show the bundling protein between two filaments

20. Actin with the bundling protein and Phalloidin Fig 12: 3D AFM height image of actin bundles Fig 11: Confocal fluorescent image of actin bundles Fig 13: AFM height image of actin bundles

21. Summary • AFM enables clear imaging of actin filaments and the binding of proteins to the filaments • Greater resolution that fluorescence microscopy • More information on the structure and size of actin filaments • The bundling protein binds to and re-organises actin filaments

22. Advantage of AFM imaging • AFM enables imaging at the molecular level, without interference of labelling molecules • Important that we can image the molecule involved in cell mechanics and investigate how they interact with each other

23. Acknowledgements • Dr. Mark Murphy • Dr. Steven Crosby • Professor David Burton • Dr. Francis Lilley • Professor Michael Lalor